Transposition, the movement of genes from one position to another within the genome, was first suggested by Barbara McClintock. These moveable genes are also knows as jumping genes, transposable elements, or transposons. They exist in all organisms (eukaryotes, prokaryotes, and viruses). Transposons are classified into two classes based on their mechanism of transposition. Retrotransposons (class 1) work by copying themselves and pasting copies back into the genome in multiple places. Initially retrotransposons copy themselves to RNA (transcription) but, in addition to being transcribed, the RNA is copied into DNA by a reverse transcriptase (often coded by the transposon itself) and inserted back into the genome. DNA transposons (class 2) do not involve an RNA intermediate. DNA fragments transpose directly from DNA segment to DNA segment: producing a DNA copy that transposes (replicative transposition or copy-and-paste mechanism) or cut/paste the transposon itself into a new locus (conservative transposition or cut- and paste mechanism) via a transposase encoded by the transposon. Maize Ac transposon, En/Spm transposon, Mutator (Mu) transposon, Drosophila P element, piggyBac transposon, and mice Sleeping Beauty (SB) transposon employ a cut-and-paste mechanism.
The maize transposon Activator (Ac) is an autonomous transposable element of 4,565 bp active in a wide range of plant species. It codes for a single gene product, Ac transposase. The transposase gene is flanked on either side by inverted repeat sequences (IR), which are essential for transposition.
The genetic modification of plants offers improvements in agricultural practices, food safety, and human health. The development of transgenic plants requires the use of selectable marker genes, because the efficiency of plant transformation is less than optimal for many important plant species. In current plant transformation systems, a selectable marker gene is co-delivered with the gene of interest (GOI) to identify and separate rare transgenic cells from non-transgenic cells. Usually, a conditional dominant gene, with no influence on the growth or morphology of plants, is used as a selectable marker. Dominant genes encoding either antibiotic or herbicide resistance are widely used as selectable markers.
The antibiotics and herbicides used to select rare transgenic cells from non-transgenic cells generally have negative effects on proliferation and differentiation. For example, glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a critical enzyme in the shikimate pathway for the biosynthesis of aromatic amino acids. Glyphosate-resistant epsps was successfully used as a selectable marker in the plant transformation of oilseed rape, soybean, potato, maize, and wheat.
Over the past few years, consumer and environmental groups have expressed concern about the use of markers from an ecological and food safety perspective. The development of marker-free transgenic plants is desirable in agricultural biotechnology. Many strategies to produce markerfree transgenic plants have been described. The Cre-lox site-specific recombination system has been widely studied for marker removal. In fact, the first markerfree commercial transgenic plant was developed using Cre-lox technology. In the transformation vector, the marker is flanked by directly oriented lox sites. A cre gene is introduced into the genome from a genetic cross. The expression of the Cre protein causes recombination between the two loxP sites, and the marker is lost during the process of recombination. New techniques have been developed to control the cre gene expression by flanked with an inducible promoter. A limitation of this system is that the high level of expression of the cre gene may result in phenotypic aberrations in some plant species (Hajdukiewicz et al. 2001). In contrast to the Cre-lox recombination system leading to the loss of the marker gene, the transposon system (e.g., Ac/Ds) offers information about the new location of the removed marker's DNA. In the transformation vector, the marker gene is inserted into the Ds element. The expression of the Ac transposase excises both ends of the transposon and usually re-integrates into other locations on the chromosome. When the transposon transposes within the same chromosome (linked transposition), both insertion sites of the T-DNA (harboring the marker gene) and the transposon (harboring the GOI) need regulatory approval for commercialization. With unlinked transposition, the marker gene can be removed by out-crossing. Although the work is time consuming, all removed information remains clear for regulatory approval. Furthermore, with the transposon system, one successful transformation can create more independent transgenic lines because of the re-integrated loci. This feature is valuable for creating transgenic plants in species with low transformation efficiency. However, out-crossing with this system cannot be used with vegetatively propagated plants and woody tree species.
US2007259430, WO0196583, US2007220627 disclose processes for removing the selectable marker gene via homologous recombination system, wherein US2007259430 and US2007220627 refer to exchange between specific recombinase recognition sequence.
U.S. Pat. No. 5,482,852 discloses biologically safe plant transformation system via crossing the transformed plant through self-crossing or with another plant to obtain F1 or more removed generation progeny; and utilizing a means for selecting those progeny that carry the gene of interest and are free of the ancillary nucleic acids.